This proposal aims to describe the basic functional characteristics of the myocardial chloride/bicarbonate exchanger, and to investigate its role in mediating increased excitability of myocardial cells during ischemia and reperfusion. Little is known about the role of anion's in myocardial homeostasis, but recent evidence has suggested that chloride and bicarbonate movements across the cardiac sarcolemma may affect arrhythmogenesis. The focus of this study will be the chloride/bicarbonate exchanger isoforms bAE3 and cAE3, which have recently been cloned from the human heart. Recombinant AE3 protein function will be studied in two heterologous expression systems, Xenopus oocytes and mammalian CHOP cells. Initially, the transport characteristics and the pH regulation of the AE3 mechanism will be defined. Subsequently, the effects of its putative physiologic activator MgATP and other possible regulatory influences such as tonicity, growth factors and second messengers will be studied. The subdomains and/or individual amino acids which mediate these influences on AE3 will be identified by chimera construction and subsequent site-directed mutagenesis. Following this initial characterization, AE3 protein will be overexpressed in rat neonatal cardiocytes to document potential consequences on pH, [Ca2+], and modulation of action potential of myocardial cells. The effects of inhibition of native AE3 function in rat neonatal cardiocytes will also be evaluated following overexpression of anti-sense AE3 RNA or introduction of anti-sense oligonucleotides. Cardiocytes which overexpress and underexpress AE3 will be studied further under conditions of simulated ischemia and reperfusion via transient exposure to acidotic medium and to metabolic inhibitors. The proposed study will define the yet unknown characteristics of the AE3 transport mechanism, and elucidate its possible role in augmented myocardial automaticity in the setting of ischemia and reperfusion. Furthermore, it will enhance our understanding of the overall role of this chloride/bicarbonate exchanger in myocardial function.